Many physiologically important events are mediated by the binding of guanine nucleotide-binding regulatory proteins (G proteins) to G protein-coupled receptors (GPCRs). These events include vasodilation, stimulation or decrease in heart rate, bronchodilation, stimulation of endocrine secretions and enhancement of gut peristalsis, development, mitogenesis, cell proliferation and oncogenesis.
G proteins are a diverse superfamily of guanine nucleotide-binding proteins that play a central role in signal transduction and regulation of cellular metabolism. They are generally comprised of three subunits: a guanyl-nucleotide binding alpha subunit; a beta subunit; and a gamma subunit. (For a review, see Conklin et al. Cell 73, 631–641, (1993)). G proteins commonly cycle between two forms, depending on whether GDP or GTP is bound to the alpha subunit. When GDP is bound, the G protein exists as a heterotrimer, the G alpha-beta-gamma complex. When an alpha-beta-gamma complex operatively associates with a ligand-activated GPCR in a cell membrane, the rate of exchange of GTP for bound GDP is increased and the G alpha subunit dissociates from the G beta-gamma complex. The free G alpha subunit and G beta-gamma complex are capable of transmitting a signal to downstream elements of a variety of signal transduction pathways, for example by binding to and activating adenyl cyclase. This fundamental scheme of events forms the basis for a multiplicity of different cell signaling phenomena.
Recent studies have suggested that all members of the GPCR superfamily have a conserved structure. Comparisons of avian and mammalian beta-adrenergic receptor cDNA's (Yarden et al., Proc. Natl. Acad. Sci. USA 83: 6795–6799, 1986; Dixon et al., Nature 321:75–79, 1986; and Kobilka et al., Proc. Natl. Acad. Sci. USA 84:46–50, 1987), a bovine rhodopsin cDNA (Nathans and Hogness, Cell 34:807–814, 1983), an alpha 2-adrenergic receptor (Kobilka et al., Science 238:650–656, 1987), an angiotensin receptor cDNA (Young et al., Cell 45:711–719, 1986; Jackson et al., Nature 335:437439, 1988), a bovine substance K receptor (Masu et al., Nature 329:836–838, 1987), and a muscarinic acetylcholine receptor cDNA (Kubo et al., Nature 323:411416, 1986) predict that all GPCR share a highly conserved presence of seven hydrophobic transmembrane domains that are suggested to be transmembrane helices of 20–30 amino acids connected by extracellular or cytoplasmic loops. Kobilka et al., Science 240: 1310 (1988); Maggio et al., FEBS Lett. 319: 195 (1993); Maggio et al., Proc. Natl. Acad. Sci USA 90: 3103 (1993); Ridge et al., Proc. Natl. Sci USA 91, 3204 (1995); Schonenberg et al., J. Biol. Chem. 270: 18000 (1995); Huang et al., J. Biol. Chem. 256: 3802 (1981); Popot et al., J. Mol. Biol. 198: 655 (1987); Kahn and Engelman, Biochemistry 31: 6144 (1992); Schoneberg et al. EMBO J. 15: 1283 (1996); Wong et al., J. Biol. Chem. 265: 6219 (1990); Monnot et al., J. Biol. Chem. 271: 1507 (1996); Gudermann et al., Annu. Rev. Neurosci. 20: 399 (1997); Osuga et al., J. Biol. Chem. 272: 25006 (1997); Lefkowitz et al., J. Biol. Chem. 263:4993–4996, 1988; Panayotou and Waterfield, Curr. Opinion Cell Biol. 1:167–176, 1989. These transmembrane domains of G-protein coupled receptors are designated TM1, TM2, TM3, TM4, TMS, TM6 and TM7. TM4, TM5, TM6 and TM7 are the most highly conserved and are postulated to provide sequences which impart biological activity to GPCRs. TM3 is also implicated in signal transduction.
The coupling of GPCRs to intracellular signaling molecules such as adenylate cyclase (Anand-Srivastava et al., J. Biol. Chem. 271: 19324–19329 (1996)) and G-proteins (Merkouris et al., Mol. Pharmacol. 50: 985–993 (1996)) is reportedly inhibited by peptides corresponding to the intracellular loops of the receptors. Those studies were conducted primarily to provide an understanding of molecular mechanisms of receptor function and could not be applied directly for drug design, because of the difficulties in intracellular delivery of the inhibitors.
WO 94/05695 and U.S. Pat. No. 5,508,384 set forth sequences of transmembrane regions for 74 GPCRs. The WO 94/05695 patent publication describes and claims polypeptides corresponding to fragments or homologous sequences of GPCRs which can bind a GPCR ligand or which can modulate ligand binding. Both references disclose that a membrane spanning fragment of the third TM domain of the dopamine D2 receptor specifically bound a ligand of the intact receptor in a simple, small unilamellar vesicle model. The fragment used was terminated with a lysine (which is positively charged at physiological pH) at one end and with an aspartic acid (which is negatively charged at physiological pH) at the other. This peptide would not be expected to insert readily into a biological membrane.